Osteoporosis, resulting from increased bone resorption relative to formation, affects millions of Americans and its prevalence is increasing with the aging population. It is estimated that half of all women over the age of 50 will have an osteoporosis-related fracture during their lifetime. The osteoclast (OC), the only cell capable of resorbing bone, is the primary therapeutic target for the treatment of osteoporosis. Thus, understanding the means by which the OC degrades bone is essential for development of new treatment options. Proper cytoskeletal organization of the OC is required for bone degradation to occur. Therefore, paxillin, a scaffolding protein essential for cytoskeletal organization in many cell types, is a candidate therapeutic target. However, the role of paxillin in OC function is not currently known. We find that while paxillin is critical to osteoclast function, its impact on the cell is counterintuitive. Specifically, the paxillin knock out (Pax-/-) OC is larger and is more sensitive to RANKL (the main activating cytokine of the OC), yet its resorptive capacity is impaired. We hypothesize that paxillin, via definable domains and/or phosphorylated residues, mediates RANKL-induced activation and cytoskeletal organization of the OC. Our specific aim is therefore to determine the mechanisms by which paxillin mediates RANKL-induced activation and cytoskeletal organization of the OC. We will accomplish this aim by assessing Rac activity, ruffled membrane formation, OC polarization, podosome organization, lamellipodia formation, migration, and adhesion in the Pax-/- OC in response to RANKL. To define the domains and phosphorylation sites on paxillin that are important in these functions, we will transduce Pax-/- embryonic stem cells with specific paxillin mutants, and again determine the functional properties as the cells differentiate into OCs in response to RANKL. As paxillin is activated by RANKL, these studies will provide insight into the mechanism by which RANKL promotes pathological bone loss, and may help elucidate new therapeutic targets for the prevention and treatment of osteoporosis.